Ophthalmic suspension composition

ABSTRACT

A suspension includes an ophthalmic active ingredient suspended in a formulation vehicle including a suspending agent and a non-ionic cellulose derivative. The ophthalmic active agent is present as particles having D V90  &lt; 5 µm and D v50 &lt; 1 µm. The suspension may be administered to a patient for treating an ophthalmic inflammatory condition.

BACKGROUND

This invention relates to an ophthalmic suspension composition,especially an ophthalmic suspension composition containing acorticosteroid that provides improved therapeutic efficacy.

Ophthalmic compositions are used to provide relief of a variety ofocular conditions and ocular disease states. Often, ophthalmiccompositions are administered or instilled to the eye via eye drops froma multi-dose container in the form of solutions, suspensions, ointmentsor gels. If the ophthalmic active component is sufficiently soluble inwater, the formulation may have the form of a solution eye drop product.However, if the solution product has too low of a viscosity, e.g., lessthan about 30 cp (or mPa s), upon instillation the ophthalmic active canbe rapidly discharged from the precorneal area of the eye because oflacrimal secretion and nasolacrimal drainage. As a result, it has beenestimated that approximately 80-99% of the ophthalmic active componentis simply washed or flushed from the eye before the active actuallycontacts the desired ocular tissue to achieve its desired clinicaleffect. The poor residence time of the active in the eye thus requiresfrequent instillation or use of a more concentrated active product toachieve the desired clinical effect. To lengthen the residence time ofophthalmic active, and thus, to enhance the bioavailability of theophthalmic active per instillation, non-solution based ophthalmicvehicles have been developed. Examples of such ophthalmic vehiclesinclude ointments, suspensions, and aqueous gels. However, theseophthalmic vehicles can have their drawbacks as well. For example, theuse of ointments often causes blurred vision just after instillation. Insome instances, the patient can sense a “goopy feeling” in their eyes,which is undesirable.

Some ophthalmic formulations have the form of the so-called in situgel-forming systems. These ophthalmic vehicles can extend precornealresidence time and improve ocular bioavailability of the ophthalmicactive. Typically, in situ gel-forming systems are aqueous solutionscontaining a polymer system. The ophthalmic products tend to exist as alow-viscosity liquid during storage in the dispenser container and forma gel upon contact with tear fluid. The liquid-to-gel transition can betriggered by a change in temperature, pH, ionic strength, or thepresence of tear proteins, depending on the particular polymer systememployed. Although a stiff gel can have an extended residence in the eyeand assist in promoting a higher drug bioavailability, and perhapsenhance clinical outcome per instillation, such in situ gel formingsystems, like the ointments, can interfere adversely with vision andresult in patient dissatisfaction. In addition, such compositions mustoften be formulated at significantly acidic pH, which is not comfortableupon installation in the eye of the patient.

In some formulations, the ophthalmic active is virtually, or completely,insoluble in an aqueous solution-based formulation. For example, U.S.Pat. Nos. 5,538,721 and 4,540,930 describe a pharmaceutical compositioncomprising an amino-substituted steroid therapeutic agent, and aneffective stabilizing amount of lightly cross-linked carboxy-containingpolymer. Cyclodextrin has also been used to at least partiallysolubilize the therapeutic agent in an aqueous medium.

Lotemax® (loteprednol etabonate (LE) ophthalmic gel, 0.5% LE) (Bausch &Lomb Incorporated) contains 5 mg/g of loteprednol etabonate, as asterile preserved ophthalmic gel suspension, and has proven effectivefor the treatment of post-operative inflammation and pain followingocular surgery. Lotemax(R) ophthalmic gel, 0.5% LE, contains boric acid,edetate disodium dihydrate, glycerin, polycarbophil, propylene glycol,sodium chloride, tyloxapol, water, and sodium hydroxide to adjust pHbetween 6 and 7, and is preserved with benzalkonium chloride (BAK)0.003%.

DUREZOL® (difluprednate ophthalmic emulsion 0.05%) (Alcon Laboratories,Inc.), a sterile preserved ophthalmic emulsion for topical ophthalmicadministration, has proven effective for the treatment of inflammationand pain associated with ocular surgery, and is also indicated for thetreatment of endogenous anterior uveitis. DUREZOL® ophthalmic emulsioncontains difluprednate (0.05%), boric acid, castor oil, glycerin, sodiumacetate, sodium EDTA, sodium hydroxide to adjust pH, polysorbate 80 andwater, and is preserved with sorbic acid 0.1%

SUMMARY OF THE INVENTION

This invention provides an ophthalmic suspension comprising anophthalmic active ingredient suspended in a formulation vehicle, whereinthe ophthalmic active ingredient is present as particles that haveD_(v90) < 5 µm and D_(v50) < 1 µm. D_(v90) is the particle diameterbelow which particles having 90% of the cumulative volume of all theparticles are present, and D_(v50) is the particle diameter below whichparticles having 50% of the cumulative volume of all the particles arepresent.

In one aspect, the active ingredient comprises an ophthalmic activepharmaceutical ingredient (“API”). In another aspect, the ophthalmic APIcomprises an anti-inflammatory agent. In still another aspect, theophthalmic API comprises a steroid (also known in the art asglucocorticosteroid or corticosteroid). In yet another aspect, theophthalmic API comprises a nonsteroidal anti-inflammatory drug(“NSAID”).

The formulation vehicle comprises a suspending agent and a non-ioniccellulose derivative. The suspending agent may comprise a carboxyvinylpolymer, such as polycarbophil or carbomer. The non-ionic cellulosederivative may be hydroxypropylmethyl cellulose.

The suspension may be storage stable for at least one year, or for atleast two years.

The ophthalmic active ingredient may be a corticosteroid, such asloteprednol etabonate or difluprednate. The active ingredient may be anon-steroid, such as nepafenac.

The formulation vehicle may further comprise a preservative and/or asurfactant.

According to various aspects, the formulation vehicle comprisespolycarbophil, hydroxypropylmethyl cellulose, benzalkonium chloride, apoloxamer surfactant, glycerin, propylene glycol, and a borate bufferagent.

The suspension may have the form of a gel at room temperature that formsa liquid upon instillation in an eye.

According to various aspects, the ophthalmic active ingredient may bepresent as particles having D_(v90) < 3 µm and D_(v50) < 1 µm, orpresent as particles having D_(v90) < 3 µm and D_(v50) < 0.6 µm, orpresent as particles having D_(v90) < 1 µm.

In another aspect, this invention provides a method of treating anophthalmic inflammatory condition that comprises administering to an eyeof a patient in need of said treating a suspension according to any ofthe aforementioned aspects. The suspension may be administered at afrequency of one or two times per day, or at a frequency of three orfour times per day. The ophthalmic inflammatory condition may beinflammation resulting from post-ocular surgery or from allergicreaction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows particle size distribution of LE samples milled in amicrofluidizer.

FIG. 2 shows change in particle size D_(v50) over time during milling ina microfluidizer.

FIG. 3 shows particle size distribution of Bead Milled vs. 30minute-Microfluidizer samples.

FIG. 4 shows particle size distribution of Bead Milled vs. 30-180minute-Microfluidizer samples.

FIG. 5 shows particle size distribution of Bead Milled samples with andwithout BAK using 2.0-, 1.0- and 0.5-mm bead diameters.

FIG. 6 shows particle size distribution of Bead Milled samples with BAKat T=0 and T=3 weeks.

FIG. 7 shows particle size distribution of Bead Milled samples withoutBAK at T=0 and T=3 weeks.

FIG. 8 shows particle size distribution of Bead Milled samples withvarying LE:Poloxamer Ratios.

FIG. 9 shows particle size distribution of Bead Milled samples withvarying LE:Poloxamer Ratios.

FIG. 10 shows particle size distribution of Bead Milled samples at 17hours vs. 34 hours.

FIG. 11 shows Individual LE Concentrations in Tear Fluid after a SingleTopical Ocular Administration to Dutch Belted Rabbits.

FIG. 12 shows Mean (± SD) LE Concentrations in Tear Fluid after a SingleTopical Ocular Administration to Dutch Belted Rabbits.

FIG. 13 shows Individual LE Concentrations in Bulbar Conjunctiva after aSingle Topical Ocular Administration to Dutch Belted Rabbits.

FIG. 14 shows Mean (± SD) LE Concentrations in Bulbar Conjunctiva aftera Single Topical Ocular Administration to Dutch Belted Rabbits.

FIG. 15 shows Individual LE Concentrations in Cornea after a SingleTopical Ocular Administration to Dutch Belted Rabbits.

FIG. 16 shows Mean (± SD) LE Concentrations in Cornea after a SingleTopical Ocular Administration to Dutch Belted Rabbits.

FIG. 17 shows Individual LE Concentrations in Aqueous Humor after aSingle Topical Ocular Administration to Dutch Belted Rabbits

FIG. 18 shows Mean (± SD) LE Concentrations in Aqueous Humor after aSingle Topical Ocular Administration to Dutch Belted Rabbits.

FIG. 19 shows Individual LE Concentrations in Iris/Ciliary Body after aSingle Topical Ocular Administration to Dutch Belted Rabbits.

FIG. 20 shows Mean (± SD) LE Concentrations in Iris/Ciliary Body after aSingle Topical Ocular Administration to Dutch Belted Rabbits.

FIG. 21 shows Mean (± SD) C_(max) and AUC_((0-24h)) (± SE) Values inTear Fluid after a Single Topical Ocular Administration to Dutch BeltedRabbits.

FIG. 22 shows Mean (± SD) C_(max) and AUC_((0-24h)) (± SE) Values inBulbar Conjunctiva after a Single Topical Ocular Administration to DutchBelted Rabbits.

FIG. 23 shows Mean (± SD) C_(max) and AUC_((0-24h)) (± SE) Values inCornea after a Single Topical Ocular Administration to Dutch BeltedRabbits.

FIG. 24 shows Mean (± SD) C_(max) and AUC_((0-24h)) (± SE) Values inAqueous Humor after a Single Topical Ocular Administration to DutchBelted Rabbits.

FIG. 25 shows Mean (± SD) C_(max) and AUC_((0-24h)) (± SE) Values inIris/Ciliary Body .after a Single Topical Ocular Administration to DutchBelted Rabbits.

FIG. 26 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Tear Fluid after a Single Topical Ocular Administration toRabbits (Groups 1 and 2).

FIG. 27 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Tear Fluid after a Single Topical Ocular Administration toRabbits (Groups 3 and 4).

FIG. 28 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Bulbar Conjunctiva after a Single Topical OcularAdministration to Rabbits (Groups 1 and 2).

FIG. 29 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Bulbar Conjunctiva after a Single Topical OcularAdministration to Rabbits (Groups 3 and 4).

FIG. 30 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Cornea after a Single Topical Ocular Administration to Rabbits(Groups 1 and 2).

FIG. 31 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Cornea after a Single Topical Ocular Administration to DutchBelted Rabbits (Groups 3 and 4).

FIG. 32 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Aqueous Humor after a Single Topical Ocular Administration toRabbits (Groups 1 and 2).

FIG. 33 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Aqueous Humor after a Single Topical Ocular Administration toRabbits (Groups 3 and 4).

FIG. 34 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Iris/Ciliary Body after a Single Topical Ocular Administrationto Rabbits (Groups 1 and 2).

FIG. 35 lists Individual and Summary Statistics for LE Concentrations(µg/g) in Iris/Ciliary Body after a Single Topical Ocular Administrationto Dutch Belted Rabbits (Groups 3 and 4).

FIGS. 36 to 38 shows Difluprednate Metabolite Concentrations in AqueousHumor after a Single Topical Ocular Administration of Difluprednate toDutch Belted Rabbits

FIG. 39 shows Difluprednate Metabolite Concentrations in Iris/Ciliaryafter a Single Topical Ocular Administration of Difluprednate to DutchBelted Rabbits

FIG. 40 shows Difluprednate Metabolite Concentrations in Cornea after aSingle Topical Ocular Administration of Difluprednate to Dutch BeltedRabbits

FIG. 41 shows Difluprednate Metabolite Concentrations in BulbarConjunctiva after a Single Topical Ocular Administration ofDifluprednate to Dutch Belted Rabbits

FIG. 42 shows Difluprednate Metabolite Concentrations in Plasma after aSingle Topical Ocular Administration of Difluprednate to Dutch BeltedRabbits

DETAILED DESCRIPTION

A variety of ophthalmic active ingredients may be employed in thisinvention. Generally, ophthalmic active ingredients include any activeingredient for the treatment of dry eye, allergy, glaucoma,inflammation, or infection.

A first class of ophthalmic active pharmaceutical ingredients (APIs) issteroids, also known in the art as glucocorticosteroids orcorticosteroids, especially for the treatment of ocular inflammatoryconditions. Examples include loteprednol etabonate, dexamethasone,fluoromethalone, prednisolone and difluprednate. Another class ofophthalmic APIs is NSAIDs, such as nepafenac. Other classes ofophthalmic APIs include anti-bacterial agents, such as besifloxacin, andimmunosuppressants, such as cyclosporine.

According to various aspects, the corticosteroid suspended in theformulation vehicle is selected from: dexamethasone at concentrations of0.1% to 0.2% by weight, fluoromethalone at concentrations of 0.05% to0.25% by weight, prednisolone at concentrations of 0.1% to 1% by weight,loteprednol etabonate at concentrations of 0.1% to 0.5% by weight anddifluprednate at concentrations of 0.01 to 0.1% by weight. Alternately,according to various aspects, the non-steroid suspending in theformulation vehicle is nepafenac at concentrations of 0.1 to 0.5% byweight.

Generally, the suspensions of the invention will include an ophthalmicactive ingredient that has a solubility in water at 25° C. and pH of 7that is less than 10% of the formulated concentration in mg/mL in theophthalmic formulation. For example, if the ophthalmic active ingredientis present in an ophthalmic formulation at a concentration of 0.1 mg/mL,the ophthalmic active will have a solubility in water at 25° C. and a pHof 7 of less than 0.1x(0.1 mg/mL), i.e., less than 0.01 mg/mL. Likewise,for an ophthalmic active that is present in an ophthalmic formulation ata concentration of 10 mg/mL, the ophthalmic active ingredient will havea solubility in water at 25° C. and a pH of 7 of less than 0.1x(10mg/mL), i.e., less than 1.0 mg/mL. Accordingly, the water solubility ofa specific agent in the suspension and the agent’s concentration in thesuspension in mg/mL are related with respect to formation of asuspension. In other words, an ophthalmic active ingredient present at arelatively high concentration in a suspension can have a somewhatgreater water solubility than another agent with a lower watersolubility present in another suspension at a lower concentration, butbecause of the higher concentration in the former suspension asignificant portion of the former agent remains suspended in theformulation.

According to various aspects, the ophthalmic active ingredient isloteprednol etabonate. Loteprednol etabonate (also referred to herein as“LE”) is a known compound and can be synthesized by methods disclosed inU.S. Pat. No. 4,996,335, the entire contents of which are herebyincorporated by reference in the present specification. According tovarious aspects, the concentration of LE in the formulation vehicle isin the range from 0.1 wt.% to 2 wt.%, or from 0.14 wt.% to 1.5 wt.%, orfrom 0.2 wt.% to 1 wt.%, or from 0.2 wt.% to 0.5 wt.%. A specificconcentration of LE may be 0.38 wt %.

Another ophthalmic active ingredient is difluprednate. Difluprednate(also referred to herein as “DFBA”) is a derivative of prednisolone anda known compound, and can be synthesized by methods known in the art.According to various aspects, the concentration of DFBA in theformulation vehicle is in the range from 0.01 to 0.1% by weight, or from0.02 to 0.07 wt. A specific concentration of DFBA may be 0.05 wt %.

The formulation vehicle includes at least one suspending agent. Oneclass of suspending agents are polymers prepared from at least about90%, or from at least about 95%, by weight, based on the total weight ofmonomers present, of one or more carboxyl-containing monoethylenicallyunsaturated monomers. Acrylic acid is a suitable carboxyl-containingmonoethylenically unsaturated monomer, but other ethylenicallyunsaturated, polymerizable carboxyl-containing monomers may be employed.These include: methacrylic acid, ethacrylic acid, β-methylacrylic acid(crotonic acid), cis-α-methylcrotonic acid (angelic acid),trans-α-methylcrotonic acid (tiglic acid), α-butylcrotonic acid,α-phenylacrylic acid, α-benzylacrylic acid, α-cyclohexylacrylic acid,β-phenylacrylic acid (cinnamic acid), coumaric acid (o-hydroxycinnamicacid), umbellic acid (p-hydroxycoumaric acid), and the like, which canbe used in addition to, or instead of, acrylic acid.

The carboxyl-containing polymers prepared from these monethylenicallyunsaturated monomers may be lightly cross-linked by employing a smallpercentage, i.e., from about 0.5% to about 5%, or from about 0.2% toabout 3%, based on the total weight of monomers present, of apolyfunctional cross-linking agent. Such cross-linking agents includingnon-polyalkenyl polyether difunctional cross-linking monomers, such as:divinyl glycol; 3,4-dihydroxy-hexa-1,5-diene;2,5-dimethyl-1,5-hexadiene; divinylbenzene; N,N-diallylacrylamide;N,N-diallylmethacrylamide; and the like.

Various lightly cross-linked polymers are commercially available, or maybe generally prepared by suspension or emulsion polymerization, usingconventional free radical polymerization catalysts. In general, suchpolymers will range in molecular weight from about 250,000 to about4,000,000, or from about 500,000 to about 2,000,000.

The lightly cross-linked polymers can be made from a carboxyl-containingmonomer or monomers as the sole monoethylenically unsaturated monomerpresent, together with the cross-linking agent or agents. They can alsobe polymers in which up to about 40%, or within the range of about 0% toabout 20% by weight, of the carboxyl-containing monoethylenicallyunsaturated monomer or monomers has been replaced by one or morenon-carboxyl-containing monoethylenically unsaturated monomerscontaining only physiologically and ophthalmologically innocuoussubstituents, including acrylic and methacrylic acid esters such asmethyl methacrylate, ethyl acrylate, butyl acrylate,2-ethylhexylacrylate, vinyl acetate, 2-hydroxyethylmethacrylate,3-hydroxypropylacrylate, and the like.

Various lightly cross-linked carboxy-containing polymers are known inthe art. For example, those disclosed in Robinson U.S. Pat. No.4,615,697, International Publication No. WO 89/06964, and Davis et alU.S. Pat. No. 5,192,535. An example of lightly cross-linked polymers areacrylic acid polymers wherein the cross-linking monomer is3,4-dihydroxyhexa-1,5-diene or 2,5-dimethylhexa- 1 ,5-diene.

Another class of lightly cross-linked polymers are carboxyl-containingpolymer prepared by suspension polymerization of acrylic acid anddivinyl glycol, including NOVEON AA-1 polycarbophil (available fromLubrizol). Other lightly cross-linked carboxy-containing polymersinclude various carbomers, such as Carbopol carbomers (available fromLubrizol). According to various aspects, the suspending agent is acarboxvinyl polymer selected from polycarbophil and carbomer.

The suspending agent serves to ensure the ophthalmic active ingredientremains in suspension in the formulation vehicle. The formulationvehicle provides a storage-stable, suspension of the ophthalmic activeingredient, such as in the form of a gel. However, once instilled intothe eye as eye drops, the gel gradually transitions to a liquid form,i.e., it loses its gel character due to the shear thinning properties ofthe gel. Following instillation, the eyelid applies shear to theformulation when the eye blinks, and this shear reduces drastically theviscosity, thereby avoiding the sticky, “goopy” sensation as found inmany ointments and eye drops intended to remain in gel form while in theeye. However, once eyelid movement ceases, thereby eliminating the shearforce, viscosity is no longer reduced, helping to maintain residence ofthe formulation on the eye. Eventually, the gel transitions entirely toliquid. In certain aspects, the ophthalmic suspension has a yield point,at which point below the composition is a solid gel, of 2-8 Pascals, andmore suitably 3-5 Pa.

The term “storage-stable” denotes that the API will remain effectivelysuspended in the formulation vehicle for an extended period of timewithout having to stir or shake the packaged composition. In otherwords, agitation of the formulation in its package is not required tore-suspend the API in the formulation vehicle. In contrast,non-storage-stable suspensions require a user to shake the packagedcomposition before instillation so that the API is uniformly distributedin the carrier vehicle; however, if the user neglects to shake thepackage, the user may not instill a consistent and proper dosage.

Accordingly, the storage-stable ophthalmic suspensions of this inventionwill consistently deliver 90% to 110% of a predetermined dosage ofpharmaceutical active per eye drop, without a patient having to agitatethe suspension in its container.

According to various aspects, the composition is storage-stable in itspackage for at least a year, in which case the shelf-life of the productis one year. According to other aspects, the composition isstorage-stable in its package for at least two years, in which case theshelf-life of the product is two years.

According to various aspects, the formulation vehicle includes anon-ionic cellulose derivative as a supplemental suspending agent.Representative agents include hydroxypropylmethyl cellulose (“HPMC”) orhydroxypropylcellulose (“HPC”).

The vehicle formulations described herein can also include various otheringredients, including but not limited to surfactants, tonicity agents,buffers, preservatives, chelating agents, co-solvents andviscosity-building agents.

Surfactants that can be used are surface-active agents that areacceptable for ophthalmic applications. Useful surface active agentsinclude polysorbate 80 (such as Tween® 80 surfactant from ICI AmericaInc), tyloxapol, and various poloxamer surfactants including poloxamer188 (such as Pluronic® F-68 surfactant available from BASF) andpoloxamer 407 (such as Pluronic® F127 available from BASF). Thesesurfactants are nonionic alkaline oxide condensates of an organiccompound which contains hydroxyl groups. The concentration in which thesurface active agent may be used is only limited by neutralization ofthe bactericidal effects on the accompanying preservatives (if present),or by concentrations which may cause eye irritation.

Various tonicity agents may be employed to adjust the tonicity of theformulation. Examples are sodium chloride, potassium chloride, magnesiumchloride, calcium chloride and nonionic diols, such as glycerol andpropylene glycol, dextrose and/or mannitol. These agents may be added tothe formulation to approximate physiological tonicity. Such an amount oftonicity agent will vary, depending on the particular agent to be added.In general, however, the formulations will have a tonicity agent in anamount sufficient to cause the final formulation to have anophthalmically acceptable osmolality (generally about 150-450 mOsm/kg).According to various aspects, a nonionic tonicity agent that alsofunctions as a demulcent may be employed.

An appropriate buffer system may be added to the formulations to preventpH drift under storage conditions. Such buffers include phosphatebuffers (e.g., sodium dihydrogen phosphate), acetate buffers (e.g.,sodium acetate), citrate buffers (e.g., sodium citrate and/or citricacid) and borate buffers (e.g., sodium borate and/or boric acid) Theparticular concentration of the buffer will vary, depending on thespecific agent employed.

Topical ophthalmic products are typically packaged in multidose form, inwhich case a preservative is generally required to prevent microbialcontamination during use. Suitable preservatives include: biguanides,hydrogen peroxide, hydrogen peroxide producers, benzalkonium chloride,chlorobutanol, benzododecinium bromide, phenylethyl alcohol, sorbicacid, polyquatemium-1, and other agents known in the art. Suchpreservatives are typically employed at a level of from 0.001 to 1%(w/w). A chelating agent, such as edetate disodium, may be included toenhance the efficacy of the antimicrobial agent used as thepreservative. In the case where the ophthalmic suspension is packaged ina unitary dose form, the sterile suspension generally does not require apreservative.

Supplemental co-solvents or viscosity-building agents may be added tothe formulation vehicles. Such materials may be included to providelubrication, to make the formulation vehicle approximate the consistencyof endogenous tears, to aid in natural tear buildup, or otherwise toprovide temporary relief of dry eye symptoms and conditions upon ocularadministration. Supplemental viscosity-building agents include polymericpolyols, such as, polyethylene glycol, dextrans such as dextran 70,water soluble proteins such as gelatin, polyvinyl alcohols,polyvinylpyrrolidones, and polysaccharides such as hyaluronic acid andits salts and chondroitin sulfate and its salts.

A representative gel suspension of this invention comprises or consistsessentially of, or consists of the following composition:

Component/Function % By Weight of Total Composition API SubmicronParticles 0.05-2% Suspending Agent 0.01-5% Non-ionic cellulosederivative 0.01-1% Preservative 0-1% Chelating Agent 0-1% Tonicity Agent0-1% Surfactant 0.01-5% Demulcent / Tonicity Agent 0.01-5% Buffer Agent0.001-2% Water as Diluent qs to 100% pH Adjuster qs to pH of 6-8

According to various aspects, a gel suspension comprises or consistsessentially of, or consists of the following composition:

Component/Function % By Weight of Total Composition Corticosteroid APISubmicron Particles 0.04-2% Carboxyvinyl Polymer Suspending Agent0.01-2% Non-ionic Cellulose Derivative 0.01-2% Preservative 0.001-1%Chelating Agent 0.01-1% Tonicity Agent 0.01-1% Nonionic Surfactant0.01-5% Nonionic Diol Demulcent/Tonicity Agent 0.01-2% Buffer Agent0.001-2% Water for injection qs to 100% Sodium hydroxide qs to pH about6.3-7.0

According to various other aspects, a gel suspension comprises orconsists essentially of, or consists of the following composition:

Component % By Weight of Total Composition Loteprednol EtabonateSubmicron Particles 0.1-0.4% Polycarbophil 0.1-0.5% HydroxypropylmethylCellulose 0.1-0.5% Benzalkonium Chloride (BAK) 0.001-0.01% EdetateDisodium Dihydrate 0.01-0.1% Sodium Chloride 0.01-0.1% PoloxamerNonionic Surfactant 0.1-1.0% Glycerin and/or Propylene Glycol 0.1-2%Borate Buffer 0.01-1% Water for injection qs to 100% Sodium hydroxide qsto pH about 6.3-7.0

A first composition, according to various aspects, comprises or consistsessentially of, or consists of the following Composition A:

Component Concentration (mg/mL) Composition A Loteprednol EtabonateSubmicron Particles 3.80 Polycarbophil, USP 3.75 HydroxypropylmethylCellulose E4M 2.50 BAK 50%^(a), EP/USP/NF 0.06 Edetate DisodiumDihydrate, USP 0.55 Sodium Chloride, USP 0.50 Poloxamer 407 2.00Glycerin, USP 8.80 Propylene glycol, USP 4.40 Boric Acid, NF 5.00 Waterfor injection qs to 1 mL Sodium Hydroxide (2N) qs to pH 6.5

(a) As BAK Solution is 50% aqueous, the final concentration in BAK is0.03 mg/mL.

According to other aspects, a gel suspension comprises or consistsessentially of, or consists of the following composition:

Component % By Weight of Total Composition Difluprednate SubmicronParticles 0.01-0.1% Carboxyvinyl Polymer Suspending Agent 0.1-0.5%Hydroxypropylmethyl Cellulose 0.1-0.5% BAK 0.001-0.01% Edetate DisodiumDihydrate 0.01-0.1% Sodium Chloride 0.01-0.1% Poloxamer NonionicSurfactant 0.01-1.0% Glycerin and/or Propylene Glycol 0.1-2% BorateBuffer 0.01-1% Water for injection qs to 100% Sodium hydroxide qs to pHabout 5.3 to 6.7

Another composition, according to various aspects, comprises or consistsessentially of, or consists of the following Composition B:

Component Concentration (mg/mL) Composition B Difluprednate SubmicronParticles 0.5 Polycarbophil, USP 3.75 Hydroxypropylmethyl Cellulose E4M2.50 BAK 50%^(a), EP/USP/NF 0.06 Edetate Disodium Dihydrate, USP 0.55Sodium Chloride, USP 0.50 Poloxamer 407 0.26 Glycerin, USP 8.80Propylene glycol, USP 4.40 Boric Acid, NF 5.00 Water for injection qs to1 mL Sodium Hydroxide (2N) qs to pH 6.0-6.5

(a) As BAK Solution is 50% aqueous, the final concentration in BAK is0.03 mg/mL.

Additional compositions, Compositions C and D, comprise, consistessentially of, or consist of:

Component Concentration (mg/mL) Concentration (mg/mL) Composition CComposition D Difluprednate Submicron Particles 0.5 0.5 Carbomer 3 3Hydroxypropylmethyl Cellulose E4M 2.5 2.5 BAK 0.03 -- Sorbic Acid 1Edetate Disodium Dihydrate, USP 0.3 0.2 Sodium Chloride, USP 0.5 0.3Poloxamer 407 0.26 0.26 Glycerin, USP 8.8 1.0 Propylene glycol, USP 4.46 Boric Acid, NF 5 1 Water for injection qs to 1 mL qs to 1 mL SodiumHydroxide (2N) qs to pH about 5.5 qs to pH about 5.5

Additional compositions, Compositions E and F, comprise, consistessentially of, or consist of:

Component Concentration (mg/mL) Composition E Composition F NepafenacSubmicron Particles 3.0 1.8 Polycarbophil, USP 3.75 3.75Hydroxypropylmethyl Cellulose E4M 2.50 2.50 BAK 50%^(a), EP/USP/NF 0.060.06 Edetate Disodium Dehydrate, USP 0.55 0.55 Sodium Chloride, USP 0.500.50 Poloxamer 407 1.53 0.92 Glycerin, USP 8.80 8.80 Propylene glycol,USP 4.40 4.40 Boric Acid, NF 5.00 5.00 Water for injection qs to 1 mL qsto 1 mL Sodium Hydroxide (2N) qs to pH 6.8 qs to pH 6.8

(a) As BAK Solution is 50% aqueous, the final concentration in BAK is0.03 mg/mL.

As mentioned, the ophthalmic suspension of this invention comprises anophthalmic active ingredient suspended in a formulation vehicle, whereinthe ophthalmic active ingredient is present as particles that haveD_(v90) < 5 µm and D_(v50) < 1 µm. D_(v90) is the particle diameterbelow which particles having 90% of the cumulative volume of all theparticles are present, and D_(v50) is the particle diameter below whichparticles having 50% of the cumulative volume of all the particles arepresent. D_(v90) and D_(v50) may be measured by light diffractiontechniques generally known in the art.

Light diffraction (LD) is a known method for determining the particlesize of materials that are suspended in a liquid or dispersed in air.The technique utilizes the principle of light diffraction whereparticles will diffract (scatter) light at angles which are inverselyproportional to their diameters. That is to say, large particles willdiffract light at small angles while small particles diffract light atlarger angles. In practice, commercially available instruments include alight source, such as a low power laser, illuminates the particlespassing through a measurement zone within a sample cell. The cone ofdiffracted light produced where the beam interacts with the particlesproduces a stationary diffraction pattern that is focused on detectors,such as two optical detector arrays. The detectors are typicallycomposed of a series of electronically separated photo-elements arrangedto measure the radial dispersion of light energy. Amount and directionof the light which strikes these detectors is electronically coded andtransmitted to a computer for processing. By taking a measurement over asuitable period of time and using a continuous flux of particles throughthe illuminated area, a representative light diffraction profile isobtained.

After measuring the diffraction pattern, the commercially availablemeasurement instruments generally include a CUP and software whichanalyzes the diffraction pattern measurement, background measurement andany required information entered by the operator (e.g., refractiveindices, particle shape, spherical or irregular) to calculate a sizedistribution model which “best fits” the observed diffraction patternprofile. Once this “best fit” is achieved, the instrument will generallyprovide a print-out or display of the size distribution parameters whichcharacterize the model. Typically, the results are given in terms of avolume size distribution. e.g., D_(v10) = x, D_(v50) = y, D_(v90) = z,etc.

For the ophthalmic suspensions of this invention, an appropriatemeasurement technique is as follows. 5 grams of the gel is weighed intoa flat bottom glass beaker. 208 grams of a 6% saline dispersant is addedto the beaker. The contents of the beaker are magnetically stirred, andan the tip of an ultrasonic processor is immersed in below the surfaceof the contents, and the contents are sonicated while the contents arestirred. Approximately a 3-mL portion of the stirred, sonicated sampleis withdrawn, and the entire portion is promptly dispensed into therecirculator bowl of the light diffraction instrument, which containsrecirculated dispersant. If necessary, additional portions of the samplesuspension may be added until reaching a transmission value of0.92-0.96. Collection of the sample diffraction pattern is initiatedwithin minutes after final addition of the sample suspension to therecirculator. The desired size distribution (e.g., D_(v10), D_(v50),D_(v90)) are generated by the instrument’s software. An example of aninstrument is the S3500 laser diffractive analyzer available fromMicrotrac (York, PA, USA and Krefeld, Germany).

Ophthalmic active ingredients with such particle sizes may be obtainedby methods generally known in the art. For example, an aqueous slurry,containing the active and the formulation vehicle, may be subjected tofluid micronization or bead milling, for a suitable time to obtain thedesired particle size. Representative techniques for fluid micronizationand bead milling are provided in Example 1. In Example 1, bead millingwas optimized to provide an ophthalmic active ingredient with thedesired particle size, but other methods, or variations of the describedbead milling methods, may be employed.

The invention will now be further described by way of several examplesthat are intended to describe but not limit the scope of the inventiondefined by the claims herein.

Example 1 - Milling of API

In the following experiments, two options for particle size reduction ofAPI were investigated: a high pressure homogenizer microfluidizer andbead milling. The high pressure homogenizer microfluidizer employed wasMicrofluidics model M-110EH microfluidizer. Additionally, variousvehicles for milling were investigated. Particle size analyses weredetermined by light diffraction (LD) unless indicated otherwise.

In a first set of experiments, studies were conducted with themicrofluidizer. As summarized in Table 1, various formulations employed10% loteprednol etabonate (LE) in 1% Polysorbate 20 (Tw20) and 0.5%boric acid with various other excipients, including Tyloxapol (Tylox)surfactant, Pluronic F68 (F68) surfactant, and benzalkonium chloride(BAK). Additionally, a formulation including 1% HPMC(hydroxypropylmethyl cellulose) and 0.2% BAK was tested. The results areshown in Table 1. It was determined from this experiment thatPolysorbate 20 was not a critical excipient for milling.

TABLE 1 Particle Size Distribution of LE Milling in Polysorbate 20Sample Vehicle Dv10 Dv50 Dv90 1% Tw20+0.1%HPMC in 0.5% Boric 0.758 1.8753.611 1%Tw20+0.5%F68 in 0.5%Boric Acid 1.248 3.332 6.419 1%Tw20+0.5%Tylox in 0.5%Boric Acid 0.642 1.543 3.016 1%Tw20+1%HPMC in0.5%Boric Acid 0.593 1.257 2.344 1%Tw20+0.07%BAK in 0.5%Boric Acid 0.5431.098 2.158 1%HPMC+0.2%BAK in 0.5%Boric Acid 0.472 0.940 2.068

In another set of experiments, four milling vehicles were tested, eachcontaining 10% LE as the API. Additionally, the milling vehiclescontained the following:

-   A -- 0.5% boric acid, 0.2% BAK, 0.5% HPMC E3-   B -- 0.5% boric acid, 0.2% BAK, 0.5% Poloxamer 407-   C -- 0.5% boric acid, 0.2% BAK, 0.5% PVP C30-   D -- 0.5% boric acid, 0.5% CMC LV; 0.2% Poloxamer 407

The samples were milled in the microfluidizer for 20 minutes in arecirculating manner at 25 k psi. Sample B, containing poloxamer 407with BAK has the narrowest and the most monomodal distribution, as seenin FIG. 1 . Sample D, containing CMC/Poloxamer, appeared larger andhighly aggregated.

In another set of experiments, API concentration was tested for itseffect on milling. Vehicles similar to Sample B above, containing 0.5%Boric acid + 0.5% Poloxamer 407 + 0.2% BAK, were employed with either10%, 20% or 30% LE. The slurries were similarly run in a recirculatingmanner in the microfluidizer at 25 k psi. Samples were taken forparticle size analysis at 10 minute, 20 minutes and 30 minute intervals.The 30-minute sample with 10% LE had suitable results. The samples withhigher concentrations of API did not appear to be more efficient. Theresults are reported in FIG. 2 .

From previous experience with bead milling, it was known that the use ofBAK during bead milling may cause aggregation. Accordingly, this wastested with LE by milling the 30% LE suspension (containing 0.5%Poloxamer 407 + 0.2% BAK) with 0.5-mm zirconium oxide (ZrO₂) beads for20 minutes in a Flacktek mixer. By light microscopy, the particle sizedistribution looked worse. This may have been due to recrystallizationas the vial became quite hot. To better control the temperature duringmilling, the sample was placed in a wrist shaker and shaken overnight.An additional sample containing 30% LE with Poloxamer 407 but no BAK wasalso placed on the wrist shaker. The particle size of both bead-milledsamples was smaller than the 30-minute microfluidizer samples(containing 0.5% Poloxamer 407 + 0.2% BAK), with no discernabledifferences for the bead-milled samples with and without BAK. The datais summarized in FIG. 3 .

To further investigate whether milling in the microfluidizer couldprovide a comparable particle size as bead milling, an additional studywas conducted using the 10% LE in the Poloxamer/BAK vehicle, with theslurries milled in the microfluidizer at 25 k psi up to 180 minutes.Samples were taken at 30-minute intervals. At 90 minutes, a particlesize of D_(v90) less than 1 µm was achieved. Additional milling timeslowly further reduced the particle size but did not yield the smallerparticle sizes achievable by bead milling. The results are reported inFIG. 4 .

Since the above experiments indicated smaller particle size was obtainedby bead milling, additional work was done to further optimize the beadmilling process.

FIG. 5 reports the particle size distribution of the followingbead-milled samples, shaken on a wrist shaker for 16 hours with theindicated ZiO₂ bead sizes:

-   A -- 20% LE, 8% poloxamer F127 -- 2.0-mm beads-   B -- 20% LE, 2% poloxamer F127 -- 1.0-mm beads-   C -- 20% LE, 2% poloxamer F127 -- 0.5-mm beads-   D -- 22% LE, 2.2% poloxamer F127, 0.225% BAK -- 0.5-mm beads-   E -- 20% LE, 2.2% poloxamer F127, 0.225% BAK -- 1.0 mm beads-   F -- 20% LE, 2.2% poloxamer F127, 0.225% BAK -- 2.0-mm beads

The smallest particle size distribution was obtained with the 0.5 mmbeads. The use of BAK in the milling slurry had no immediate effect onthe particle size.

FIG. 6 reports particle size distribution of bead-milled slurriescontaining 30% LE, poloxamer 407 and BAK, and FIG. 7 reports particlesize distribution of bead-milled slurries containing 30% LE andpoloxamer 407 but no BAK, at Time=0 and after three weeks (T=3 wks). TheBAK sample (FIG. 6 ) showed particle growth, whereas there was nosignificant change in the slurry without BAK (FIG. 7 ).

Additionally, the API:Surfactant ratio was studied. Samples were madecontaining 30% LE + 0.714% Boric acid + Poloxamer 407 (Pluronic F127surfactant) to obtain LE:Poloxamer ratios of 20:1, 10:1 and 5:1. Thesamples were milled with 0.5-mm ZiO₂ beads on a wrist shaker overnight.The results are summarized in FIG. 8 . The lower poloxamer concentrationshowed better results than the higher concentrations (noting thiscontradicts information from a literature article (Liu et al.,“Nanosuspensions of poorly soluble drugs: preparation and development bywet milling”, Int. J. of Pharm 411(1-2):215-222, 2011).

Additional 30% LE samples were made using 30:1, 40:1 and 50:1LE:Poloxamer ratios. The 20:1, 30:1 and 40:1 samples showed similarparticles sizes after 17 hours of milling (FIG. 9 ). These three sampleswere then milled for an additional 17 hours. After a total of 34 hoursmilling, the 40:1 sample became larger and bimodal possibly due to aninsufficient amount of surfactant for the increase in surface area ofthe LE. Both the 30:1 and 20:1 samples continued to get smaller overtime, with the 20:1 sample obtaining the smallest particle size (FIG. 10).

Example 2 -- Stabilization of Polycarbophil Formulation

The present inventors recognized that sub-micron particles of LE in apolycarbophil formulation are not physically stable and tend toaggregate over time. It is believed the polycarbophil polymer forms anopen mesh type of structure that produces a shear thinning gel butallows unimpeded movement of particles within the matrix. In contrast,hydroxypropylmethyl cellulose (HPMC) forms a more compact structure thatcan enhance the viscosity within the polycarbophil matrix reducingparticle movement. Also, HPMC inhibits nucleation stabilizing smallparticles by reducing the Oswald ripening effect. Accordingly, thisstudy was designed to understand the contribution of viscosityenhancement and nucleation inhibition on the stabilizing property ofhydroxypropylmethyl cellulose (HPMC), especially HPMC E4M, and otherpotential stabilizers.

Various samples of 0.38% LE gel were made employing polycarbophil alongwith 0.25% HPMC E4M or other stabilizers. Slurries were bead-milled asin Example 1. These samples were then placed in glass vials andincubated at 25° C. and 40° C. At various time points the samples wereremoved and tested for particle size by the light diffraction technique.The particle sizes after 8.5 months, stored at 40° C., are reported inTable 2: VMD denotes volume mean diameter, and D_(v95) denotes theparticle diameter below which particles having 95% of the cumulativevolume of all particles fall. Table 2 summarizes the stabilizers thatfunctioned to enhance viscosity and/or inhibit nucleation.

TABLE 2 Stabilizer Viscosity Enhancer Nucleation inhibitor VMD Dv95 P0.25% HPMC E4M X X 0.94 3.99 P 0.15% HPMC E4M X X 0.87 3.23 MP 0.05%HPMC E4M X X 1.23 3.48 NP 0.0006% HPMC E4M X X 2.68 12.33 NP NoStabilizer 3.45 22.24 MP 0.25% HPMC E3LV X 1.15 3.62 NP 0.25% CMC X 3.3319.47 NP 0.25% PVP X 3.89 29.23 P 0.25% Soluplus X 0.83 3.61 P =Protected MP = Moderately Protected NP = Unprotected

Titration of HPMC E4M shows a minimum critical concentration ofapproximately 0.05% was needed to stabilize the sub-micron LE particles.The HPMC E3LV sample has similar nucleation inhibition properties asHPMC E4M but provides lower viscosity enhancement, and shows moderateprotection similar to the 0.05% HPMC E4M sample. The polyvinylpyrrolidinone (PVP) sample provides viscosity enhancement and exhibitsresults similar to the no-stabilizer sample. Carboxymethyl cellulose(CMC) is a known suspending agent providing both viscosity enhancementand surface activity. The grade used here has low viscosity. This samplealso exhibits results similar to the no-stabilizer sample. Soluplus™stabilizer (available from BASF), containing a polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, is astrong nucleation inhibitor with no viscosity enhancement. This sampleshows stability similar to the 0.25% HPMC E4M sample.

This study shows the ideal combination of viscosity enhancement andnucleation inhibition was provided by HPMC, especially grade E4M, andthis supplemental suspending agent was effective to stabilize thesub-micron particles.

Example 3 -- Rheology Study

The effectiveness of topical corticosteroids can be limited by theirdissolution and residence time on the ocular surface. This studyexamined the dissolution and viscoelastic characteristics of CompositionA (containing submicron LE particles, 0.38%) as compared to commercialLE gel, Lotemax® Ophthalmic Gel, 0.5%.

Yield stress and oscillatory rheology measurements were performed usinga TA Instruments rheometer fitted with a vane rotor and cup containing40 g undiluted product at 25° C. Dissolution of LE submicron particles(0.6-µm diameter) and LE micronized particles (3-µm diameter used inLotemax® Ophthalmic Gel, 0.5%) was measured at 200% of saturation inPBS/0.45% SDS using a VanKel dissolution tester. Dissolution was alsodetermined in a flow-through assay simulating tear flow on the eye. An8-mL LE submicron or micronized suspension was mixed with 3 mL ofPBS/3.75% BAK. PBS/3.75% BAK was then flowed through the diluted LEsuspension at 10 mL/min. Samples were taken from the outflow and theamount of dissolved LE was determined by HPLC. This method simulates an11-µL tear volume with a flow rate of 10 µL/min.

Rheology analysis of submicron LE gel 0.38% shows a yield stress ofapproximately 4 Pa, confirming the gel structure is similar to Lotemax®Ophthalmic Gel, 0.5%. There was a 2.6-fold increase in dissolution withthe submicron LE (0.38%) as compared to micron LE (0.5%) at 30 sec. The0.38% submicron sample reached saturation at approximately 1.5 minutescompared to approximately 5 minutes for the 0.5 micronized sample. Inthe flow-through model there was an increase in dissolution over alonger time period for the submicron vs. micronized LE. Comparison ofthe AUC of the concentration vs. time curve at increasing drugconcentration indicated that there was a 1.3 fold increase overall inthe rate of dissolution with the 0.38% submicron vs. the 0.5% micronizedformulation.

Accordingly, the submicron LE gel, 0.38% (Composition A) has similarviscoelastic characteristics as Lotemax® Ophthalmic Gel 0.5%, andtherefore was expected to be storage-stable, non-settling, and provideuniform drug delivery from the container.

Example 4 -- Investigation of the Effect of Particle Size andConcentration on the Ocular Pharmacokinetics of Loteprednol EtabonateFollowing a Single Topical Ocular Administration to Dutch Belted Rabbits

The purpose of this study was to assess the effect of particle size onthe ocular pharmacokinetics (PK) of loteprednol etabonate (LE) followinga single topical ocular administration to Dutch Belted rabbits, anddetermine if a higher concentration of LE provides increased ocularexposure. Lotemax® Gel (Loteprednol etabonate 0.5% ophthalmic gel) (LE)is a potent corticosteroid in a polycarbophil-based gel formulationapproved for the treatment of post-operative pain and inflammationfollowing ocular surgery. This investigation was designed to evaluatethe effect of reduced particle size on the ocular PK of LE following asingle, topical ocular administration to Dutch Belted rabbits. A thirdformulation that contained the same sized particles of LE as thecommercial formulation but at a higher concentration was also evaluatedto determine if a higher dose of LE provides increased ocular exposure.Lotemax® Gel was used as the comparator formulation.

A total of 108 male Dutch Belted rabbits were used in this non-GLP,non-crossover pharmacokinetic study. The rabbits were approximately 7-8months of age and weighing between 1.56-2.69 kg. Prior to the start ofthe study, animals were randomly assigned to one of four study groups.On the day of dosing, animals received a single 35-µL topical oculardose containing the appropriate formulation into each eye.

-   Animals in Group 1 received a 0.38% gel formulation prepared with    submicron-sized particles of LE (Formulation 1)-   Animals in Group 2 received a 0.38% gel formulation that contained    micronized particles of LE (Formulation 2)-   Animals in Group 3 received a 0.75% gel formulation that had the    same particle size as the current Lotemax® Gel product (Formulation    3).-   Animals in Group 4 received the marketed product Lotemax® Gel (0.5%)    (Formulation 4).

Animals were observed throughout the duration of the study for generalhealth and appearance. At pre-determined time intervals after dosing,animals were euthanized and selected ocular tissue samples werecollected. Concentrations of LE in ocular tissues were determined byLC/MS/MS. All in-life procedures were conducted at PharmOptima (Portage,MI). Bioanalysis of ocular tissue samples was conducted at Bausch + Lomb(Rochester, NY). The experimental formulations were prepared by Bausch +Lomb Formulations Development and shipped to the test site as ready touse materials. Lotemax® Gel was provided by Bausch + Lomb (Tampa, FL).

Details of the test formulations are provided in Table 4-1.

TABLE 4-1 Loteprednol Etabonate Formulation Summary Concentration(mg/mL) -- Balance Water Component Formulation 1: Submicron ParticleSize Lot #3463EP026-3 Formulation 2: Micronized Particles Lot#3463EP026-2 Formulation 3: Unmodified Particle Size Lot# 3463EP026-1Formulation 4: Lotemax Gel Lot# 2420850307 Disodium EDTA 0.55 0.55 0.550.55 Sodium Chloride 0.5 0.5 0.5 0.5 Polycarbophil 3.75 3.75 3.75 3.75Tyloxapol 0.5 0.5 0.5 0.5 BAK 30 ppm 30 ppm 30 ppm 30 ppm Glycerin 8.88.8 8.8 8.8 Propylene Glycol 4.4 4.4 4.4 4.4 Boric Acid 5.0 5.0 5.0 5.0Sodium Hydroxide q.s. to pH 6.5 q.s. to pH 6.5 q.s. to pH 6.5 q.s. to pH6.5 Loteprednol Etabonate 3.90 3.96 7.44 5 API Lot # AAX-009A EQ3011 lot7289 XA21010 lot 071279545 172121 Particle Size (D_(V50)) 0.6 µm 2.87 µm2.67 µm 2.71 µm

An ophthalmic examination was performed on both eyes of all studyanimals prior to shipment from the vendor to the test facility. Theexamination consisted of an evaluation of the anterior segment of theeye using a slit lamp binocular microscope to verify that there were nopre-existing ophthalmic abnormalities that would interfere with theoutcome of the study. Upon arrival at the test facility, a visualexamination was performed on all animals to confirm that they were ingood health. Animals were then weighed and randomly assigned to one offour study groups of 27 animals each using a random number generator.

On the day of dosing, animals (fed) received a single, 35-µL topicalocular administration of the appropriate test formulation into each eye.Animals in Group 1 received a 0.38% gel formulation prepared withsubmicron-sized particles of LE (also referred to as SubmicronFormulation and Formulation). Animals in Group 2 received a 0.38% gelformulation that contained micronized particles of LE (also referred toas Micronized Formulation and Formulation 2). Animals in Group 3received a 0.75% gel formulation that had the same particle size as thecurrent Lotemax® Gel product (also referred to as Unmodified Formulationand Formulation 3), and animals in Group 4 received Lotemax® Gel (0.5%)(also referred to as Comparator and Formulation 4). The formulationswere not shaken prior to administration. Doses were instilled into thelower conjunctival sac of each eye using a calibrated Gilson M-50positive displacement pipette. Immediately after dosing, the eyelidswere gently held closed for several seconds to facilitate evendistribution of the test substance over the surface of the eye and tominimize runoff. Animals were observed throughout the duration of thestudy for general health and appearance.

At pre-determined time intervals after dosing, animals (n =3/group/collection time) were humanely euthanized by intravenousoverdose of sodium pentobarbital and ocular tissues were collected fromeach eye. Tear fluid (collected using Schirmer tear strips), bulbarconjunctiva, and aqueous humor (collected using a needle and syringe)were collected in situ, while the cornea and iris/ciliary body werecollected once the eyes had been enucleated and flash-frozen in liquidnitrogen. Ocular tissue samples were collected at 0.0833 (5 min), 0.25(15 min), 0.5 (30 min), 1, 2, 4, 8, 12, and 24 hours after dosing.Ocular tissue samples were stored frozen until being shipped on dry iceto Bausch+Lomb facility. Upon arrival, the samples were maintained at orbelow -20° C. until bioanalysis.

Concentrations of LE in ocular tissues were determined by LC/MS/MS. Forthe purpose of calculating mean concentrations, a value of ½ the lowerlimit of quantitation (LLQ) was assigned to all samples withconcentrations below the LLQ (BLQ). In addition, any sample with ameasured concentration that was BLQ and at least 10-fold below themedian concentration or more than 10-fold above the median concentrationin the respective sample pool was considered an outlier, and notincluded in any calculations. Based on these criteria, 17 (~8%) tearfluid samples, 3 (~1%) bulbar conjunctiva samples, 2 (~1%) corneasamples, 3 (~1%) aqueous humor samples, and 1 (~0.5%) iris/ciliary bodysample were determined to be outliers.

Pharmacokinetic analysis of the composite concentration vs. time datawas performed using non-compartmental methods in WinNonlin Professional®(version 5.3, Pharsight Corporation, St. Louis, MO). Due to thedestructive nature of the sampling regimen employed in this study, meancomposite data were used in the PK analysis. Nominal sample collectiontimes were used in the PK analysis. PK parameters including maximumconcentration (C_(max)) and the time at which the maximum concentrationwas observed (T_(max)) were determined directly from the concentrationvs. time profiles. The area under the concentration vs. time curve(AUC_((0-24h))) values and the corresponding standard error (SE)estimates were calculated using the linear trapezoid method in WinNonlinand/or Microsoft Excel (2010).

To determine if exposure to LE in ocular tissues followingadministration of the experimental formulations (Formulations 1-3)varied significantly from exposure obtained with the commercial product(Formulation 4), the (AUC_((0-24h))) and SE estimates were comparedusing Welch’s t-test as demonstrated by Schoenwald (1987) and Tang-Liuand Burke (1988). Schoenwald RD, Harris RG, Turner D, et al. Ophthalmicbioequivalence of steroid/antibiotic combination formulations. BiopharmDrug Dispos. 1987; 8:527-548; Tang-Liu DD, Burke PJ. The effect of azoneon ocular levobunolol absorption: calculating the area under the curveand its standard error using tissue sampling compartments. Pharm Res.1988; 5:238-241. A two-tailed Student’s t-test was used to determinesignificant differences in C_(max) after an F-test was used to determineequal or unequal variance between individual concentration values atC_(max). Differences were considered statistically significant when thecalculated P value was less than or equal to 0.05. All statisticalcalculations were performed using Microsoft Excel (2010).

The pharmacokinetic parameter values obtained for LE following a singlebilateral topical ocular administration are presented in Table 4-2. Meanand individual concentration vs. time data are presented in FIGS. 11through 25 , where BLQ denotes below level of quantification. In FIGS.21-24 , the asterisk (^(∗)) denotes statistically significant (p≤0.05)from Lotemax® Gel 0.5% LE. A summary of mean and individualconcentration vs. time data is presented in FIGS. 26-35 ; in thesefigures, the superscript (a) denotes an apparent outlier, individualresult differed by > 10-fold from other results at this collection time,so value was not included in calculations. In FIGS. 33 and 34 , asuperscript (b) denotes a result below the lower limit of quantitation,so the value equal to ½ the value of the LLQ was assigned for thepurpose of calculating summary statistics.

TABLE 4-2 Pharmacokinetic Parameter Values for Loteprednol EtabonateFollowing a Single Topical Ocular Administration to Dutch Belted RabbitsDose Group Tissue/Matrix C_(max) (µg/g) Tmax (h) AUC_((0-24h)) (µg*h/g)Group 1: Submicron Formulation 0.38% (3.8 mg/mL) (133 µg/eye) Tear fluid614 ± 691 0.0833 260 ± 49.2 Bulbar Conjunctiva 12.0 ± 12.7 0.0833 33.5 ±4.30 Cornea 3.29 ± 1.13 0.0833 6.93 ± 0.798 Aqueous Humor 0.0281 ±0.00665 1 0.0421 ± 0.00247^(a) Iris/Ciliary Body 0.165 ± 0.0793 0.250.338 ± 0.0314 Group 2: Micronized Formulation 0.38% (3.8 mg/mL) (133µg/eye) Tear fluid 201 ± 269 0.0833 157 ± 26.4 Bulbar Conjunctiva 78.7 ±102 0.25 55.0 ± 10.6 Cornea 2.22 + 1.01 0.25 3.61 ± 0.436 Aqueous Humor0.0135 ± 0.00313 0.5 0.0183 ± 0.00107 Iris/Ciliary Body 0.126 ± 0.07580.25 0.299 ± 0.0335 Group 3: Unmodified Formulation 0.75% (7.5 mg/mL)(262.5 µg/eye) Tear fluid 673 ± 1020 0.25 384 ± 101^(a) BulbarConjunctiva 22.4 ± 31.0 0.25 96.6 ± 18.0 Cornea 2.59 ± 1.20 0.0833 8.38± 1.43 Aqueous Humor 0.0190 ± 0.0273 0.25 0.0282 ± 0.00382 Iris/CiliaryBody 0.255 ± 0.311 0.25 0.491±0.0586 Group 4: Lotemax Gel 0.5% (5 mg/mL)(175 µg/eye) Tear fluid 871 ± 942 0.25 483 ± 96.6^(a) Bulbar Conjunctiva16.4 ± 19.7 0.25 95.0 ± 16.7 Cornea 2.61 ± 1.13 0.0833 6.66 ± 0.672Aqueous Humor 0.0112 ± 0.00586 0.5 0.0228 ± 0.00349 Iris/Ciliary Body0.102 + 0.118 0.0833 0.385 ± 0.0841 C_(max): Maximum mean (± SD)concentration observed after dosing; T_(max): time C_(max) was observed;AUC_((0-24h)): Mean (± SE) area under the concentration versus timecurve from the time of dosing through 24 hours. (^(a)) AUC and/orstandard error (SE) estimates calculated in Excel (reported) varyslightly from values obtained in WinNonlin due to rounding differences.Note: For aqueous humor, the relevant units for C_(max) and AUC areµg/mL and µg^(∗)h/mL, respectively.

Bioanalytical Method Summary -- The LC/MS/MS methods for thequantitation of loteprednol etabonate (LE) in Dutch Belted rabbit oculartissues were developed at Bausch + Lomb. The methods were assessed forprecision and accuracy, but were not fully validated or GLP-compliant.Overall, the performance of the analytical methods was deemed acceptableto support this nonclinical PK study.

A total of 1080 ocular tissue samples were successfully analyzed in 6bioanalytical runs. Samples consisted of tear fluid, bulbar conjunctiva,cornea, aqueous humor, and iris/ciliary body. A variable amount of 1:1acetonitrile: water was added to the tear fluid, bulbar conjunctiva,cornea, and iris/ciliary body samples using a Tecan Freedom EVO 150. Thevolume of solvent was adjusted for each sample based on the individualsample weight to ensure a constant matrix concentration for all samples,standards, and quality control samples. All samples were sonicated andvortexed prior to transferring an aliquot to a 96-well sample plate. Allsamples above the HLQ were diluted 100X with 1:1 acetonitrile: water.Two sets of at least 8 standards and 3 quality controls (low, mid, andhigh, in triplicate) along with two ‘zero’ samples (blank matrix withinternal standard) and 5 control blanks (blank matrix) were included ineach bioanalytical run.

A summary of the bioanalytical range for Dutch Belted rabbit oculartissues is provided in Table 4-3.

TABLE 4-3 Bioanalytical Range Summary Matrix Ave. Tissue Weight (mg)Assay Range of Standard Curve^(a) LLQ^(b) ULQ^(b,c) Max. Dilution TearFluid 5.11 0.1-1000 ng/mL 12.5 ng/g 12500000 ng/g 100 Bulbar Conjunctiva57.2 0.1-1000 ng/mL 1.45 ng/g 1450000 ng/g 100 Cornea 68.0 0.1-1000ng/mL 1.22 ng/g 12200 ng/g 1 Aqueous Humor NA 0.1-1000 ng/mL 0.100 ng/mL1000 ng/mL 1 Iris/Ciliary Body 86.2 0.1-1000 ng/mL 0.962 ng/g 9620 ng/g1 Abbreviations: LLQ - lower limit of quantitation ULQ - upper limit ofquantitation NA - not applicable ^(a) Nominal concentration (ng/mL) ofanalyte in 1:1 acetonitrile: water ^(b) Approximate limits ofquantitation based on average tissue weights. ^(c) ULQ includes amaximum sample dilution factor used.

Discussion

With the exception of the tear fluid, exposure to LE was similar orgreater in all ocular tissues examined following administration of thehigher (0.75%) concentration unmodified LE formulation (Formulation 3)compared with Lotemax® Gel (Formulation 4). In most cases, the observeddifferences in exposure were less-than-proportional to dose (1.0- to2.5-fold difference based on C_(max) and AUC_((0-24h))) and notstatistically significant.

When compared to Lotemax® Gel, exposure to LE following administrationof the 0.38% micronized formulation (Formulation 2) was 1.2- to 4.3-foldlower in all ocular tissues examined, based on C_(max) andAUC_((0-24h)). In the tear fluid and cornea, the differences in exposurewere statistically significant.

Topical ocular administration of the lower concentration (0.38%)formulation prepared with submicron particles of LE (Formulation 1)provided significantly (p ≤ 0.05) greater exposure to LE in the aqueoushumor (1.9- to 2.5-fold), and similar or slightly greater exposure to LEin the iris/ciliary body (1.0 - to 1.6-fold) and cornea (1.0 to1.3-fold) compared with Lotemax® Gel (Formulation 4), based on C_(max)and AUC_((0-24h)) values. Exposure to LE was lower in the tear fluid(1.4- to 1.9-fold) and significantly lower in the bulbar conjunctiva(1.4- to 2.8-fold), but these are not considered target tissues. Insummary, based on C_(max) and/or AUC₍₀₋ _(24h)) values, exposure to LEwas significantly greater in the aqueous humor, similar or greater inthe iris/ciliary body and cornea, and lower in the tear fluid and bulbarconjunctiva following administration of the 0.38% submicron formulation(Formulation 1) compared to Lotemax® Gel. Despite the 24% reduction inadministered dose, exposure to LE was statistically higher in theaqueous humor, a key target tissue, for the 0.38% submicron formulation(Formulation 1). This data indicates that the submicron particle sizeenhances drug penetration to key ocular tissues.

The fold differences for the formulations, as compared to Lotemax® Gel0.5% LE, are summarized in Tables 4-4 to 4-6.

TABLE 4.4 -- Fold Differences -- Formulation 1 (0.38% LE Submicron) vs.Formulation 4 (0.5% Lotemax Gel) C_(max) AUC_((0-24h)) C_(max)Significant AUC Significant Tear Fluid 0.70 0.54 No No BulbarConjunctiva 0.73 0.35 No Yes Cornea 1.26 1.04 No No Aqueous Humor 2.511.85 Yes Yes Iris/Ciliary Body 1.62 1.01 No No

TABLE 4.5 -- Fold Differences -- Formulation 2 (0.38% LE Micronized) vs.Formulation 4 (0.5% Lotemax Gel) C_(max) AUC_((0-24h)) C_(max)Significant AUC Significant Tear Fluid 0.23 0.33 No Yes BulbarConjunctiva 4.80 0.58 No No Cornea 0.85 0.54 No Yes Aqueous Humor 1.210.80 No No Iris/Ciliary Body 1.24 0.78 No No

TABLE 4.6 Fold Differences -- Formulation 3 (0.75% LE Unmodified) vs.Formulation 4 (0.5% Lotemax Gel) C_(max) AUC_((0-24h)) C_(max)Significant AUC Significant Tear Fluid 0.77 0.80 No No BulbarConjunctiva 1.37 1.02 No No Cornea 0.99 1.26 No No Aqueous Humor 1.701.24 No No Iris/Ciliary Body 2.50 1.28 No No

Example 5 -- Investigation of the Effect of Particle Size andConcentration on the Ocular and Systemic Pharmacokinetics of21-Desacetyl Difluprednate Following a Single Topical OcularAdministration of Difluprednate in a Gel Formulation to Dutch BeltedRabbits

A total of 135 Dutch Belted rabbits were used in this pharmacokineticstudy. On the day of dosing, animals received a single 35-µL topicalocular dose containing the appropriate formulation into each eye.Difluprednate (DFBA) is a prodrug and is rapidly hydrolyzed to21-desacetyl DFBA after ocular instillation. The LC/MS/MS methods forthe quantitation of 21-desacetyl DFBA in Dutch Belted rabbit oculartissues and plasma were assessed for precision and accuracy, but werenot fully validated or GLP-compliant. Overall, the performance of theanalytical methods was deemed acceptable to support this nonclinical PKstudy.

-   Formulation 5-1 -- 0.05% Submicron DFBA Gel Suspension --    corresponding to Composition B with D_(v50) of 0.2 µm and pH    6.1-6.2.-   Formulation 5-2 -- 0.2% Submicron DFBA Gel Suspension -- Similar to    Formulation 5-1 but containing 0.2% DFBA-   Formulation 5-3 -- 0.8% Submicron DFBA Gel Suspension -- Similar to    Formulation 5-1 but containing 0.8% DFBA-   Formulation 5-4 -- 0.8% Micronized DFBA Gel Suspension -- Similar to    Formulation 5-3 but containing 0.8% DFBA with Dv50 of 3 µm-   Formulation 5-5 -- DUREZOL® difluprednate ophthalmic emulsion, 0.05%    DFBA, a sterile preserved ophthalmic emulsion

FIGS. 36-42 report the concentrations of the DFBA metabolite,21-desacetyl difluprednate, in various ocular tissues and plasma. Table5.1 summarizes the effect of formulation and particle size of DFBA onthe exposure of the metabolite in rabbit aqueous humor after singletopical ocular administration of DFBA. Table 5.2 summarizes the effectof formulation and particle size of DFBA on the systemic exposure of21-desacetyl DFBA in plasma after single topical ocular administrationof DFBA.

TABLE 5.1 The effect of formulation and particle size on the exposure of21-desacetyl DFBA in aqueous humor after a single topical ocularadministration of DFBA to rabbits PK Parameter Form. 5-5 Form. 5-1 Form.5-2 Form. 5-3 Form. 5-4 C_(max)(ng/mL) 60.3 185 314 350 377 T_(max) (h)0.5 0.5 0.5 1.0 0.5 AUC_((0-last)) (ng·h/mL) 143 425 603 830 880

TABLE 5.2 The effect of formulation and particle size on the systemicexposure of 21-desacetyl DFBA after a single topical ocularadministration of DFBA to rabbits PK Parameter Form. 5-5 Form. 5-1 Form.5-2 Form. 5-3 Form. 5-4 C_(max)(ng/mL) 1.4 4.57 8.21 10.9 4.25 T_(max)(h) 0.25 0.25 0.25 0.5 0.25 AUC_((0-last)) (ng·h/mL) 0.761 8.67 18.721.7 11.3

The following observations were made from this study.

A single topical ocular administration of 0.05% DFBA with a submicronparticle size gel suspension (Formulation 5-1) led to significantlygreater concentrations and exposure of the active metabolite in allocular tissues, compared to the 0.05% DFBA commercial emulsion product(Formulation 5-5). C_(max) and AUC increased approximately 3-fold inaqueous humor.

The greatest concentration of DFBA metabolite concentration and exposurewere found in the conjunctiva and cornea, followed by iris/ciliary bodyand aqueous humor.

Administering increasing concentrations of DFBA in a submicron gelformulation led to an increase in the exposure of the active metabolitein aqueous humor and other ocular tissues; however, this increase wasnot dose-proportional. There was no significant differences in C_(max)in aqueous humor between submicron gel formulations at increasingconcentrations.

There was no significant difference between submicron and micronizedparticles at 0.8% DFBA (Formulations 5-3 and 5-4) in any ocular tissues,except iris/ciliary body.

Systemic exposure of Formulation 5-1 was significantly greater aftertopical administration as compared to the Formulation 5-5. Systemicexposure also increased with increasing concentrations of DFBA(Formulations 5-2, 5-3 and 5-4).

Example 6 - Stability Studies

Stability studies have shown that Composition A is storage-stable for atwo-year shelf-life.

This invention has been described by reference to certain preferredembodiments; however, it should be understood that it may be embodied inother specific forms or variations thereof without departing from itsspecial or essential characteristics. The embodiments described aboveare, therefore, considered to be illustrative in all respects and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description.

What is claimed is:
 1. An ophthalmic suspension comprising an ophthalmicactive ingredient suspended in a formulation vehicle, wherein theophthalmic active ingredient is present as particles that have D_(v90) <5 µm and D_(V50) < 1 µm, and the formulation vehicle comprises asuspending agent and a non-ionic cellulose derivative.
 2. The suspensionof claim 1, which is storage stable for at least one year.
 3. Thesuspension of claim 1, which is storage stable for at least two years.4. The suspension of claim 1, wherein the ophthalmic active ingredientis a corticosteroid.
 5. The suspension of claim 4, wherein theophthalmic active ingredient is loteprednol etabonate.
 6. The suspensionof claim 4, wherein the ophthalmic active ingredient is difluprednate.7. The suspension of claim 1, wherein the suspending agent comprises acarboxyvinyl polymer.
 8. The suspension of claim 7, wherein thecarboxyvinyl polymer is selected from the group consisting ofpolycarbophil and carbomer.
 9. The suspension of claim 8, wherein thenon-ionic cellulose derivative is hydroxypropylmethyl cellulose.
 10. Thesuspension of claim 9, wherein the non-ionic cellulose derivative ishydroxypropylmethyl cellulose E4M.
 11. The suspension of claim 1,wherein the formulation vehicle further comprises a surfactant.
 12. Thesuspension of claim 11, wherein the surfactant comprises poloxamer 407.13. The suspension of claim 1, wherein the formulation vehicle comprisespolycarbophil, hydroxypropylmethyl cellulose, a poloxamer surfactant,glycerin, propylene glycol, and a borate buffer agent.
 14. Thesuspension of claim 1, comprising loteprednol at 0.1 wt% to 0.4 wt%. 15.The suspension of claim 14, comprising loteprednol etabonate at 0.38wt%.
 16. The suspension of claim 11, comprising difluprednate at 0.01 to0.1 wt%.
 17. The suspension of claim 16, comprising difluprednate at0.05 wt%.
 18. The suspension of claim 1, wherein the ophthalmic activeingredient is present as particles having D_(v90) < 3 µm and D_(v50) < 1µm.
 19. The suspension of claim 1, wherein the ophthalmic activeingredient is present as particles having D_(v90) < 3 µm and D_(v50) <0.6 µm.
 20. A method of treating an ophthalmic inflammatory conditioncomprising administering to an eye of a patient in need of said treatinga suspension comprising an ophthalmic anti-inflammatory activeingredient suspended in a formulation vehicle, wherein the ophthalmicanti-inflammatory active ingredient is present as particles that haveD_(v90) < 5 µm and D_(v50) < 1 µm, and the formulation vehicle comprisesa suspending agent and a non-ionic cellulose derivative..
 21. The methodof claim 20, wherein the suspension is administered at a frequency ofone or two times per day.
 22. The method of claim 20, wherein theophthalmic inflammatory condition is inflammation resulting frompost-ocular surgery or from uveitis.
 23. The method of claim 20, whereinthe ophthalmic active ingredient is a corticosteroid and the formulationvehicle comprises polycarbophil, hydroxypropylmethyl cellulose,benzalkonium chloride, a non-ionic surfactant, glycerin, propyleneglycol, and a borate buffer agent.